1. Field of the Invention
The human serum contains at least three known binders of Vitamin B-12, namely the three transcobalamins designated as TC-I, TC-II, and TC-III. TC-I and TC-III are derived from granulocytes and both are alpha-globulins with a similar molecular weight, of about 120,000. They have a different electrical charge and hence differ in their electrophoretic mobility. TC-II is a beta-globulin of molecular weight of about 38,000 and it is derived mainly from the liver. The physiological functions of the three transcobalamins are not fully understood, but it is known that endogenous Vitamin B-12 is bound mainly to TC-I (about 85%), and TC-II binds about 15% of endogenous B-12 while TC-III seems to bind Vitamin B-12 only in vitro. Since TC-II binds small quantities of endogenous B-12 while it takes up the main part of Vitamin B-12 added to the serum in vitro, most of the unsaturated B-12 binding sites are located on TC-II (unsaturated B-12 binding capacity, UBBC). Vitamin B-12 is bound in the serum to the transcobalamins in a 1:1 molar ratio.
It is well known that certain pathological conditions are associated with significant specific changes in the level of the three transcobalamins in serum and that the determination of the Vitamin B-12 binding capacity of each of the three transcobalamins is an important tool in medical diagnosis. Amongst others, the quantitative determination of the B-12 binding capacity of the three transcobalamins is of value in the effective screening of certain malignant diseases and also in the monitoring of the treatment of these diseases. Amongst others, the determination of three transcobalamins is of value in:
A. diagnosis, evaluation of treatment and monitoring of the course of myeloproliferative diseases [CML (chronic myelocytic leukemia), APL (acute promyeolocytic leukemia), polycythemia vera.] PA0 B. differentiation of leukemoid reactions and conditions manifested by non-leukemic leukocytosis. PA0 C. recognition of rapid malignant cell proliferation in lymphoma, sarcoma, Hodgkins Disease, acute leukemia, etc. PA0 D. evaluation of therapy and monitoring the course of malignant diseases (remission and relapse) such as sarcomas, acute leukemias, Hodgkins Disease, lymphomas etc. PA0 E. diagnosis and recognition of hepato-cellular damage. PA0 a. DEAE-cellulose chromatography to separate TC-I from TC-II and TC-III and a Sephadex G-200 column to further separate TC-II from TC-III; PA0 b. Adsorption of TC-II on charcoal and subsequent separation of TC-I and TC-III by a DEAE-cellulose chromatography; PA0 c. Selective removal of TC-II from serum by Quso G-32 (a microfine precipitated silica) and subsequent separation of TC-I from TC-III on DEAE-cellulose; PA0 d. Separation of TC-II on a G-200 column and subsequent separation of TC-I from TC-III by a DEAE-cellulose column. PA0 e. Selective removal of TC-II by precipitation with ammonium sulfate and further separation of TC-I and TC-III from each other by DEAE-cellulose chromatography.
The quantitative determination of B-12 binding capacity of the three transcobalamins may also be of value in the recognition, differentiation and monitoring of various other disorders.
2. Description of the Prior Art
The three transcobalamins present in human serum are difficult to separate and their quantitative determination is both complicated and time-consuming. The main problem is the similarity of electrophoretic properties of TC-II and TC-III and their similar behaviour on DEAE-cellulose separation.
The present determinations require at least two steps, namely:
The above mentioned two-step procedures are rather laborious and require from two to three days to complete. Thus these are actually tools of a research laboratory and indeed the known procedures have not gained widespread acceptance as routine laboratory method in clinical laboratories.